JC virus (JCV) causes a fatal demyelinating disease in AIDS patients known as progressive multifocal leukoencephalopathy (PML). This investigation was undertaken to understand the role of alternative T antigen splicing in the regulation of JCV replication in PML brain and the influence of HIV-1 on such regulation. An intron-differential RNA PCR was developed to study the expression of alternatively spliced JCV early mRNAs in PML patients with and without AIDS. The method utilizes primers which span the large T and small t antigen introns allowing amplification of specific cDNAs in the presence of contaminating viral genomic DNA. Hybridization with junctional probes and DNA sequence analysis confirmed the identity of the PCR products. The results show that JCV early mRNA is alternatively spliced as previously predicted by analogy to SV40. Large T antigen mRNA was detected in all the brain tissues from PML patients with and without AIDS. The expression of small t antigen mRNA varied depending upon the association of PML with AIDS and upon other unknown factors. Of the 12 PML/AIDS brain tissues, 11 (92%) expressed small t antigen mRNA, whereas only 8 of 13 (62%) with PML alone showed detectable levels of small t antigen mRNA. HIV-1 proviral DNA was detected in 10 of 12 PML-AIDS brains. The results indicate that alternative splicing of JCV early mRNA is regulated in the human brain, and that the production of small t antigen may not be essential for the pathogenesis of PML.